Antibodies that Inhibit Transport Activity of Peptide Transporters

ABSTRACT

The present inventors discovered that the PepT-binding antibodies have the ability to inhibit the transport activity of peptide transporters, as a result of dedicated research. These antibodies may be utilized as cell growth inhibitors, for example, for treating and preventing cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/550,987, filedMay 25, 2006, which is a National Stage of International Application No.PCT/JP2004/004331, filed Mar. 26, 2004, which claims the benefit ofInternational Application No. PCT/JP2003/03975, filed on Mar. 28, 2003,and Japanese Patent Application Serial No. 2003-110898, filed on Apr.15, 2003. The contents of all of the foregoing applications are herebyincorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to antibodies that inhibit the transportactivity of peptide transporters, and to cell growth inhibitorscomprising the antibodies as an active ingredient.

BACKGROUND ART

Mammalian animals need to incorporate nutrients from an external source,and many transport proteins are known to exist in cells. Peptidetransporters (peptide transport proteins) transport peptides, and manyhave been found to date (for example, Non-Patent Documents 1, 2, and 3;and Patent Documents 1, 2, and 3). Peptide transporters can beclassified into proteins that import peptides into cells, and proteinsthat export peptides from cells. They can also be classified by thedifference of the energy sources used in transport. Proton-drivenpeptide transporters, which carry out transport by utilizing thedifference in proton concentrations between the inside and outside of acell, belong to the PTR family (Non-Patent Document 3). Peptidetransporters that carry out transport using ATP in the body belong tothe ABC family (Non-Patent Document 4).

It has been reported that peptide transporters are involved in thetransport of not only small-molecule peptides such as dipeptides andtripeptides, but also pharmaceutical agents such as β-lactam antibioticsand ACE inhibitors (Non-Patent Documents 5, 6, 7, 8, 9, and 10).

PepT1 and PepT2 are proton-driven peptide transporters which contributeto the absorption of proteins and the maintenance of peptidic nitrogensources through uptake of small-molecule peptides into cells. PepT1 andPepT2 are 12-transmembrane proteins, consisting of 708 and 729 aminoacids, respectively (Non-Patent Documents 1, 2, and 11).

PepT1 and PepT2 have also been reported to transport pharmaceuticalssuch as β-lactam antibiotics and bestatin (Non-Patent Documents 12, 13,and 14).

PepT1 is mainly expressed in the small intestine, and its expression inkidney and pancreas has also been confirmed. PepT2 expression has beenconfirmed in kidney, brain, lung, and spleen. PepT1 and PepT2 have beenreported to be localized in the small intestine, as well as in the brushborder membrane of renal tubular epithelial cells (Non-Patent Documents15, 16, 17, and 11).

Furthermore, overexpression of PepT1 in the cell membranes of humanpancreatic ductal carcinoma cell lines (Non-Patent Document 18), andPepT2 mRNA expression in human pancreatic ductal carcinoma cell lines(Non-Patent Document 19) have been reported. However, the involvement ofPepT1 and PepT2 in cancer cell growth is not clear, and the question ofwhether inhibiting the functions of PepT1 and PepT2 will affect cancercell proliferation has never been discussed.

[Patent Document 1] Japanese Patent Application Kokai Publication No.(JP-A) H6-261761 (unexamined, published Japanese patent application)

[Patent Document 2] JP-A H11-172

[Patent Document 3] U.S. Pat. No. 5,849,525[Non-Patent Document 1] J. Biol. Chem., 270(12):6456-6463, (1995)[Non-Patent Document 2] Biochim. Biophys. Acta., 1235:461-466, (1995)[Non-Patent Document 3] Mol. Microbiol., Vol. 16, p 825, (1995)[Non-Patent Document 4] Annu. Rev. Cell. Biol., Vol. 8, p 67, (1992)[Non-Patent Document 5] Ganaphthy, Leibach., Curr. Biol. 3, 695-701,(1991)[Non-Patent Document 6] Nakashima et al., Biochem. Pharm. 33, 3345-3352,(1984)[Non-Patent Document 7] Friedman, Amidon., Pharm. Res., 6, 1043-1047,(1989)[Non-Patent Document 8] Okano et al., J. Biol. Chem., 261, 14130-14134,(1986)[Non-Patent Document 9] Muranushi et al., Pharm. Res., 6, 308-312,(1989)[Non-Patent Document 10] Friedman, Amidon., J. Control. Rel., 13,141-146, (1990)[Non-Patent Document 11] Terada, Inui, Tanpakusitsu Kakusan Kouso(Proteins, Nucleic acids, Enzymes), Vol. 46, No. 5, (2001)[Non-Patent Document 12] Saito, H. et al., J. Pharmacol. Exp. Ther.,275, 1631-1637, (1995)[Non-Patent Document 13] Saito, H. et al., Biochim. Biophys. Acta.,1280, 173-177, (1996)[Non-Patent Document 14] Terada, T. et al., J. Pharmacol. Exp. Ther.,281, 1415-1421 (1997)[Non-Patent Document 15] Ogihara, H. et al., Biochem. Biophys. Res.Commun 220, 848-852, (1996)[Non-Patent Document 16] Takahashi, K. et al., J. Pharmacol. Exp. Ther.,286, 1037-1042 (1998)[Non-Patent Document 17] Hong, S. et al., Am. J. Physiol. Renal.Physiol., 276, F658-F665 (1999)

[Non-Patent Document 18] Cancer Res., 58, 519-525, (1998) [Non-PatentDocument 19] Millennium World Congress of Pharmaceutical Sciences,(2000) DISCLOSURE OF THE INVENTION

The present invention was made in view of such circumstances.Specifically, an objective of the present invention is to provideantibodies that inhibit the transport activity of peptide transporters,and cell growth inhibitors comprising the antibodies as an activeingredient, particularly, cell growth inhibitors for cancers such aspancreatic cancer.

The present inventors discovered that substances which inhibit thetransport activity of peptide transporters suppress cell growth.Furthermore, the present inventors discovered antibodies that inhibitthe transport activity of peptide transporters. These findings show thatcell growth can be suppressed by inhibiting the activity of peptidetransporters using the antibodies. Suppression of peptide transporteractivity is considered as an important indicator in the development ofgrowth inhibitors against cancer cells and such.

More specifically, the present invention provides:

(1) an antibody that has ability to inhibit the transport activity of apeptide transporter;(2) the antibody of (1), wherein the peptide transporter is PepT1 orPepT2;(3) the antibody of (2), wherein the peptide transporter is PepT1;(4) the antibody of any one of (1) to (3), wherein the antibody is amonoclonal antibody;(5) a cell growth inhibitor that comprises the antibody of any one of(1) to (4) as an active ingredient;(6) an anti-cancer agent that comprises the antibody of any one of (1)to (4) as an active ingredient;(7) the anti-cancer agent of (6), wherein the cancer is pancreaticcancer;(8) a method for inhibiting the transport activity of a peptidetransporter, wherein the method comprises the step of contacting a cellwhich expresses the peptide transporter with an antibody that binds tothe peptide transporter;(9) the method of (8), wherein the peptide transporter is PepT1 orPepT2;(10) the method of (9), wherein the peptide transporter is PepT1;(11) a method for suppressing cell growth, wherein the method comprisesthe step of inhibiting the transport activity of a peptide transporterby contacting a cell that expresses the peptide transporter with anantibody that binds to the peptide transporter;(12) the method of (11), wherein the peptide transporter is PepT1 orPepT2;(13) the method of (12), wherein the peptide transporter is PepT1;(14) the method of any one of (11) to (13), wherein the cell is a cancercell; and,(15) the method of (14), wherein the cancer cell is a pancreatic cancercell.

The present invention provides antibodies having the ability to inhibitthe transport activity of peptide transporters. Peptide transporters ofthe present invention are not particularly limited; however, they arepreferably peptide transporters which incorporate peptides into cellsusing proton motive force. More preferably, they are PepT1 or PepT2, andmost preferably, they are PepT1.

The nucleotide and amino acid sequences of PepT1 and PepT2 are alreadyknown (human PepT1: GenBank XM_(—)007063 (J. Biol. Chem.,270(12):6456-6463, (1995)); and human PepT2: GenBank XM_(—)002922(Biochim. Biophys. Acta., 1235:461-466, (1995))).

The antibodies of the present invention having the ability to inhibitthe transport activity of peptide transporters are not particularlylimited, as long as they can inhibit peptide transporter-mediatedtransport (for example, peptide transporter-mediated peptide uptake intocells). Peptide transporter-mediated transport inhibition does notrequire complete blockage of peptide transport. A decrease in the amountof peptide transported would be sufficient.

There are no limitations on the antibodies of the present invention, aslong as they can bind to peptide transporters and inhibit the transportactivities of the peptide transporters. Mouse antibodies, ratantibodies, rabbit antibodies, sheep antibodies, camel antibodies,chimeric antibodies, humanized antibodies, human antibodies, and suchmay be suitably used. The antibodies may be polyclonal or monoclonal,but monoclonal antibodies are preferred in view of the stable productionof homogeneous antibodies. Polyclonal antibodies and monoclonalantibodies can be produced by methods well known to those skilled in theart.

Hybridomas that produce monoclonal antibodies can be prepared as followsusing basically conventional techniques. Specifically, the hybridomascan be prepared by (1) conducting immunization using a desired antigen,or cells expressing a desired antigen, as the sensitizing antigenaccording to normal immunization methods; (2) fusing the obtainedimmunized cells with conventional parent cells by normal cell fusionmethods; and (3) screening for monoclonal antibody-producing cells(hybridomas) using normal screening methods. For example, mammaliananimals such as mice, rats, rabbits, sheep, and monkeys can be used asanimals to be immunized. Antigens can be prepared according toconventional methods such as methods using baculoviruses (e.g., WO98/46777). When peptide transporters expressed on a baculovirus membraneare used as an immunogen, gp64 transgenic mice may be used as theimmunized animal (International Patent Application No. WO 03/104453).

Hybridomas can be produced, for example, according to the method ofMilstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981)73: 3-46). When the antigen has low immunogenicity, immunization can beperformed by linking it to a macromolecule with immunogenicity, such asalbumin.

Recombinant antibodies can also be used, which can be produced by (1)cloning an antibody gene from a hybridoma; (2) incorporating theantibody gene into an appropriate vector; (3) introducing the vectorinto a host; and (4) producing the recombinant antibodies by geneticengineering techniques (see, for example, Carl, A. K. Borrebaeck, James,W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the UnitedKingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNAs of thevariable regions (V regions) of antibodies are synthesized fromhybridoma mRNAs using reverse transcriptase. When DNAs encoding a Vregion of an antibody of interest are obtained, they are linked to DNAsencoding an antibody constant region (C region) of interest, and thenincorporated into expression vectors. Alternatively, DNAs encoding anantibody V region can be incorporated into expression vectors comprisingDNAs of an antibody C region. The DNAs are incorporated into expressionvectors such that expression is controlled by expression regulatoryregions such as enhancers and promoters. Host cells are then transformedwith these expression vectors to express the antibodies.

In the present invention, recombinant antibodies artificially modifiedto reduce xenoantigenicity against humans can be used. Examples of suchinclude chimeric antibodies and humanized antibodies. These modifiedantibodies can be produced using known methods. A chimeric antibody isan antibody comprising the antibody heavy chain and light chain variableregions of a nonhuman mammal such as a mouse, and the antibody heavychain and light chain constant regions of a human. A chimeric antibodycan be obtained by (1) ligating the DNA encoding a variable region of amouse antibody to the DNA encoding a constant region of a humanantibody; (2) incorporating them into an expression vector; and (3)introducing the vector into a host for production of the antibody.

A humanized antibody, which is also called a reshaped human antibody, isobtained by transplanting a complementarity determining region (CDR) ofan antibody of a nonhuman mammal such as a mouse, into the CDR of ahuman antibody. Conventional genetic recombination techniques for thepreparation of such antibodies are known. Specifically, a DNA sequencedesigned to ligate a CDR of a mouse antibody with the framework regions(FRs) of a human antibody is synthesized by PCR, using severaloligonucleotides constructed to comprise overlapping portions at theirends. A humanized antibody can be obtained by (1) ligating the resultingDNA to a DNA which encodes a human antibody constant region; (2)incorporating this into an expression vector; and (3) transfecting thevector into a host to produce the antibody (see, European PatentApplication No. EP 239,400, and International Patent Application No. WO96/02576). Human antibody FRs that are ligated via the CDR are selectedwhen the CDR forms a favorable antigen-binding site. As necessary, aminoacids in the framework region of an antibody variable region may besubstituted such that the CDR of a reshaped human antibody forms anappropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993)53, 851-856).

Those skilled in the art can determine the CDRs by well-knowntechniques, for example, using the database of antibody amino acidsequences prepared by Kabat et al. (“Sequences of Proteins ofImmunological Interest” U.S. Dept. Health and Human Services, 1983) toanalyze the homology.

Methods for obtaining human antibodies are also known. For example,desired human antibodies retaining antigen-binding activity can beobtained by (1) sensitizing human lymphocytes in vitro with antigens ofinterest or cells expressing antigens of interest; and (2) fusing thesensitized lymphocytes with human myeloma cells such as U266 (seeExamined Published Japanese Patent Application No. (JP-B) Hei 1-59878).Alternatively, a desired human antibody can also be obtained by using adesired antigen to immunize a transgenic animal that comprises theentire repertoire of human antibody genes (see International PatentApplication WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO96/34096, and WO 96/33735). Furthermore, techniques to obtain humanantibodies by panning a human antibody library are known. For example,the variable region of a human antibody is expressed as a single chainantibody (scFv) on the surface of a phage using phage display method,and phages that bind to the antigen can be selected. By analyzing thegenes of selected phages, the DNA sequences encoding the variableregions of human antibodies that bind to the antigen can be determinedIf the DNA sequences of scFvs that bind to the antigen are known,appropriate expression vectors to which these sequences are inserted canbe constructed to obtain human antibodies. Such methods are well known(see WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO95/01438, and WO 95/15388).

When the antibody genes have been isolated and introduced into anappropriate host, hosts and expression vectors can be used inappropriate combination to produce the antibodies. As eukaryotic hostcells, animal cells, plant cells, and fungal cells may be used. Knownanimal cells include (1) mammalian cells such as CHO, COS, myeloma, babyhamster kidney (BHK), HeLa, and Vero cells; (2) amphibian cells such asXenopus oocytes; or (3) insect cells such as sf9, sf21, and Tn5. Knownplant cells include cells derived from the Nicotiana genus such asNicotiana tabacum, which can be cultured as callus. Known fungal cellsinclude yeasts such as the Saccharomyces genus, for exampleSaccharomyces cerevisiae, and filamentous fungi such as the Aspergillusgenus, for example Aspergillus niger. Prokaryotic cells can also be usedin production systems that utilize bacterial cells. Known bacterialcells include E. coli and Bacillus subtilis. By transferring theantibody genes of interest into these cells using transformation, andthen culturing the transformed cells in vitro, the antibodies can beobtained.

Furthermore, the antibody may be an antibody fragment or a modifiedantibody thereof, as long as it binds to PepT and inhibits its function.For example, the antibody fragment may be Fab, F(ab′)2, Fv, or singlechain Fv (scFv) or a diabody, in which Fv from H or L chains are ligatedby an appropriate linker. More specifically, the antibody fragment isobtained by (1) treating the antibody with enzymes such as papain andpepsin; (2) transferring it into an expression vector; and then (3)expressing it in an appropriate host cell (see, for example, Co, M. S.et al., J. Immunol. (1994) 152, 2968-2976; Better, M. & Horwitz, A. H.Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.;Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496,Academic Press, Inc.; Lamoyi, E., Methods in Enzymology (1989) 121,663-669; and Bird, R. E. et al., TIBTECH (1991) 9, 132-137). scFv can beobtained by ligating the V regions of the antibody H-chain and L-chain.In scFv, the V regions of the H chain and L chain are ligated via alinker, preferably via a peptide linker (Huston, J. S. et al., Proc.Natl. Acad. Sci. U.S.A (1988) 85, 5879-5883). The V regions of the scFvH chain and L chain may be derived from any of the antibodies describedherein. The peptide linker used to ligate the V regions may be, forexample, any single-chain peptide consisting of 12 to 19 residues. DNAencoding scFv can be amplified by PCR using as a template either wholeDNA, or a partial DNA encoding a desired DNA, selected from a DNAencoding the H chain or the V region of the H chain of the aboveantibody, and a DNA encoding the L chain or the V region of the L chainof the above antibody; and using a primer pair that defines the bothends. Further amplification can be subsequently conducted using thecombination of DNA encoding the peptide linker portion, and the primerpair that defines both ends of the DNA to be ligated to the H chain andthe L chain respectively. Once DNAs encoding scFvs are constructed,expression vectors containing the DNAs, and hosts transformed by theseexpression vectors, can be obtained according to conventional methods.Furthermore, scFvs can be obtained according to conventional methodsusing the resulting hosts. These antibody fragments can be produced inhosts by obtaining genes encoding the antibody fragments and expressingthem in a manner similar to that outlined above. Diabodies are dimersformed by linking two fragments (such as scFvs; hereinafter referred toas diabody-constituting fragments), in which one variable region islinked to the other variable region via a linker or such. Ordinarily,diabodies comprise two VLs and two VHs (Holliger, P. et al., Proc. Natl.Acad. Sci. USA, 90, 6444-6448 (1993); EP 404097; WO 93/11161; Johnson etal., Method in Enzymology, 203, 88-98, (1991); Holliger et al., ProteinEngineering, 9, 299-305, (1996); Perisic et al., Structure, 2,1217-1226, (1994); John et al., Protein Engineering, 12(7), 597-604,(1999); Holliger et al., Proc. Natl. Acad. Sci. USA., 90, 6444-6448,(1993); Atwell et cd., Mol. Immunol. 33, 1301-1312, (1996)).

Antibodies bound to various molecules such as polyethylene glycol (PEG)may be used as modified antibodies. Furthermore, cytotoxic substancessuch as radioisotopes, chemotherapeutic agents, and bacteria-derivedtoxins may be attached to the antibodies. Such modified antibodies canbe obtained by chemically modifying the obtained antibodies. Methods forantibody modification have been established in the art.

Antibodies used in the present invention may be bispecific antibodies.Bispecific antibodies may be those that comprise antigen binding siteswhich recognize different epitopes of a peptide transporter molecule.Alternatively, one antigen binding site may recognize a peptidetransporter, while another antigen binding site may recognize acytotoxic substance, such as a radioactive substance, chemotherapeuticagent, or cell-derived toxin. Bispecific antibodies can be produced byjoining the HL pairs from two types of antibodies, or by fusinghybridomas that produce different monoclonal antibodies to generatebispecific antibody-producing fusion cells. Furthermore, bispecificantibodies can be produced by genetic engineering techniques.

Alternatively, antibodies with modified sugar chains may also be used inthe present invention. Techniques for modifying antibody sugar chainsare already known (for example, WO 00/61739, WO 02/31140). The“antibodies” in the present invention include such antibodies.

Antibodies expressed and produced as described above can be purified byconventional methods for purifying normal proteins. Antibodies can beseparated and purified by, for example, appropriately selecting and/orcombining affinity columns such as a protein A column, or achromatography column, filtration, ultrafiltration, salt precipitation,dialysis, and such (Antibodies A Laboratory Manual. Ed Harlow, DavidLane, Cold Spring Harbor Laboratory, 1988).

Conventional means can be used to measure the antigen-binding activityof the antibodies (Antibodies A Laboratory Manual. Ed Harlow, DavidLane, Cold Spring Harbor Laboratory, 1988). For example, an enzymelinked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), aradioimmunoassay (RIA), or a fluoroimmunoassay may be used.

Whether a particular molecule binds to a peptide transporter can bedetermined using conventional methods. Examples of conventional methodsinclude immunoprecipitation, West-Western blotting, ELISA, EIA, RIA,fluoroimmunoassay, and methods using a biosensor utilizing surfaceplasmon resonance effect.

Whether antibodies inhibit the transport function of peptidetransporters can be determined using known methods, for example, bylabeling substrates such as peptides, with a radioactive substance(e.g., ¹⁴C), u fluorescent substance, or such, and then measuring theamount of substrate uptake into the peptide transporter-expressing cells(International Patent Application No. WO 03/083116, Zoku Iyakuhin noKaihatsu 4-Yakubutsu no Seitaimakuyusou to Soshikihyoutekika I, II(Development of Pharmaceuticals 4—Transport of Pharmaceuticals ThroughBiological Membranes and Tissue Targetting I, II) (Terada, Hiroshi,Tsuji, Akira. et al. ed).

There are no particular limitations on the cells to be targeted by thecell growth inhibitors of the present invention, but cancer cells suchas pancreatic cancer cells, liver cancer cells, lung cancer cells,esophageal cancer cells, breast cancer cells, and colon cancer cells arepreferred, and pancreatic cancer cells are especially preferred. Thecell growth inhibitors of the present invention are used for the purposeof treating and preventing diseases caused by cell growth, and morespecifically cancers such as pancreatic cancer.

The antibodies of the present invention may be utilized not only asinhibitors of peptide transporter-mediated transport, but also as cellgrowth inhibitors, because they are capable of suppressing cell growthas shown in the Reference Examples. The cell growth inhibitors can beadministered either orally or parenterally, but are preferablyadministered parenterally. Specific examples include injections,transnasal administrations, transpulmonary administrations, andtransdermal administrations. For example, injections can be administeredsystemically or locally by intravenous injection, intramuscularinjection, intraperitoneal injection, or subcutaneous injection.Furthermore, the method of administration can be selected appropriatelyaccording to the age and symptoms of the patient. A single dose can beselected, for example, from within the range of 0.0001 mg to 1,000 mgper kg body weight. Alternatively, the dose can be selected, forexample, from within the range of 0.001 to 100,000 mg per patient.However, the dose of a therapeutic agent of the present invention is notlimited to these examples. Furthermore, the therapeutic agents of thepresent invention can be formulated according to standard methods (see,for example, Remington's Pharmaceutical Science, latest edition, MarkPublishing Company, Easton, U.S.A.), and may comprise pharmaceuticallyacceptable carriers and additives.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of detecting the inhibition ofPepT1 activity by anti-human PepT1 monoclonal antibodies inPepT1-expressing viruses. The PepT1 activity on viral membranes wasmeasured as the amount of [¹⁴C] glycylsarcosine uptake by the viruses.The data are shown as an average±S.D. (n=3 to 4).

FIG. 2 is a graph showing the PepT1 inhibiting ability of AT-264 in BaF3cells.

FIG. 3 is a graph showing the cell growth inhibitory effect of AT-264against human pancreatic cancer cell line AsPC-1. The data are shown asan average±S.D. (n=3 to 4).

FIG. 4 is a graph showing the PepT2 inhibiting ability of AT-264 inBaF3/PepT2. The data are shown as an average±S.D. (n=3 to 4).

FIG. 5 is a graph showing the cell growth inhibitory effect of AT-264against human pancreatic cancer cell line BxPC-3. The data are shown asan average±S.D. (n=6).

FIG. 6 shows the nucleotide sequence of the gp64 gene construct.

FIG. 7 is a continuation of FIG. 6.

FIG. 8 shows the structure of the pCAG-gp64 vector construct.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is specifically illustrated withreference to Examples, but it is not to be construed as being limitedthereto.

Example 1 Production of anti-PepT1 Antibodies Using gp64 Transgenic Mice(TgM)

Primary immunization was performed by subcutaneously injecting a PBSsuspension containing PepT1-BV equivalent to a 1 mg protein and 200 ngof pertussis toxin into gp64 TgM (International Patent Application No.WO 03/104453). Subsequent immunizations were performed by subcutaneouslyinjecting PepT1-BV equivalent to a 500 μg protein (not containing thepertussis toxin) prepared in a similar manner. The final immunizationwas performed by injecting PepT1-BV equivalent to a 250 μg protein viathe tail vein. Spleen cells were prepared from these mice and were fusedwith mouse P3U1 cells by a standard polyethylene glycol method.Screening was performed using FACS with BaF/3-PepT1 cells. Furthermore,monoclonal antibodies (clones 113, 119, and 253) which specifically bindto PepT1 were established with a FACS using BaF/3-PepT2 cells.

Example 2 Determining the Ability to Inhibit PepT1 Transport Activity

[¹⁴C] Glycylsarcosine was diluted with HBSS (pH6.0) to a finalconcentration of 50 μM to prepare a substrate solution. Mouse monoclonalantibodies (clones 119, 253, and 113), which recognize the extracellularregion of human PepT1, were diluted with PBS to a final concentration of200 μg/mL to prepare antibody solutions. 20 μL of a solution of buddingbaculovirus expressing PepT1(50 μg protein), the N terminus of which isattached to a His-tag, and 20 μL of each of the antibody solutions weremixed, and pre-incubated at 37° C. for one hour. 160 μL of the substratesolution pre-warmed to 37° C. was added to the virus solution toinitiate the reaction. One minute later, 1 mL of ice-cold HBSS (pH7.4)(hereinafter referred to as “quenching solution”) was added to stop thereaction. The virus-containing reaction solution was immediatelyfiltered by suction through a mixed cellulose membrane filter, and thenwashed twice with 5 mL of the quenching solution. The membrane filterswere transferred into liquid scintillation vials, and 5 mL of Clear-solI was added to dissolve the filters. After dissolution, theradioactivity on the filters was measured on a liquid scintillationcounter. To measure non-specific adhesion to the filter, the quenchingsolution was added in a similar procedure before the substrate solutionwas added, and the measured value was subtracted from each of the valuesobtained from the experiments described above.

The inhibition of PepT1 activity by anti-human PepT1 monoclonalantibodies is shown in FIG. 1. As a control, the PepT1 activity in theabsence of the antibodies was indicated as 100. Among the three types ofanti-human PepT1 monoclonal antibodies, clone 119 inhibits approximately20% of the PepT1 activity, and clone 253 inhibits approximately 10% ofthe activity in comparison to the control. The PepT1 activity inhibitionwas statistically significant (Student t-test). The above revealed thatanti-PepT1 antibodies may have the ability to inhibit PepT1 transportactivity.

SEQ ID NO: 1 represents the amino acid sequence of the heavy-chainvariable region of clone 119.

Reference Example 1 AT-264's Inhibitory Effect on PepT1 Activity

AT-264 has a structure represented by the structural formula below. Thefollowing experiment confirmed that the compound is a peptidetransporter (PepT) inhibitor.

The ability of AT-264 to inhibit PepT1 was examined by forcedlyexpressing human PepT1 in mouse bone marrow-derived BaF3 cells(hereinafter abbreviated as BaF3/PepT1). The results showed that theuptake of the radioactive substrate [¹⁴C] glycylsarcosine into cells wasinhibited in a concentration-dependent manner (FIG. 2). Accordingly,AT-264 was found to inhibit PepT1 functions.

Reference Example 2 The Cell Growth Inhibitory Effect of AT-264 AgainstHuman Pancreatic Cancer Cell Line AsPC-1

AT-264 was dissolved in RPMI1640-10 mM Hepes (hereinafter, abbreviatedas ‘the medium’) containing 0.5% ethanol and 0.5% DMSO to prepare 2.5 mMAT-264 solution. Then, this solution was diluted with the medium toprepare 0.625 mM and 0.0625 mM AT-264 solutions.

A 5×10⁴ cells/mL solution of human pancreatic cancer cell line AsPC-1was prepared using a medium containing 50% FBS. This suspension wasseeded at 40 μL/well (2×10³ cells) onto a 96-well plate pre-coated withCollagen type I, and 160 μL of the AT-264 solution was added. This wascultured for six days in a CO₂ incubator (on the second day of culture,100 units/mL penicillin and 0.1 mg/mL streptomycin were added). On thesixth day of culture, the number of viable cells was quantified by MTSassay.

The results of the cell growth experiment are shown in FIG. 3. Cellgrowth inhibition was confirmed to be approximately 30% in the presenceof 2 mM AT-264, and was present even in the presence of 0.5 mM AT-264,although slight. Morphological changes to AsPC-1 were not observed underthe microscope, even in the presence of AT-264. Furthermore, the resultsof RT-PCR indicated that in AsPC-1, PepT1 expression was greater thanPepT2. Accordingly, cell growth inhibition by AT-264 was considered tobe due to the inhibition of PepT1 function, and not to non-specificcytotoxicity.

Reference Example 3 Inhibitory effect of AT-264 on PepT2 Activity

The ability of AT-264 to inhibit PepT2 was examined using a murine bonemarrow-derived cell line BaF3 in which human PepT2 is forcedly expressed(hereinafter, abbreviated as BaF3/PepT2). As a result, the uptake of theradioactive substrate [³H] glycylsarcosine into cells was inhibited in aconcentration-dependent manner (FIG. 4). Accordingly, AT-264 was foundto inhibit the function of not only PepT1, but also of PepT2.

Reference Example 4 Cell Growth Inhibitory Effect of AT-264 on HumanPancreatic Cancer Cell Line BxPC-3

AT-264 was dissolved in RPMI1640-10 mM Hepes with 100 units/mLpenicillin and 0.1 mg/mL streptomycin (hereinafter, abbreviated as ‘themedium’) containing 0.5% ethanol and 0.5% DMSO to prepare 2.5 mM AT-264solution. Furthermore, this solution was diluted with the medium toprepare 0.625 mM and 0.0625 mM AT-264 solutions.

5×10⁴ cells/mL solution of BxPC-3 was prepared using the mediumcontaining 50% FBS. This suspension was seeded at 40 μL/well (2×10³cells) onto a 96-well plate pre-coated with Collagen type I, and 160 μLof AT-264 solution was added. This was cultured for six days in a CO₂incubator, and on the sixth day of culture, the number of viable cellswas quantified by MTS assay.

The results of the cell growth experiment are shown in FIG. 5. Cellgrowth inhibition was confirmed to be approximately 75% in the presenceof 2 mM AT-264, and approximately 20% even in the presence of 0.5 mMAT-264. Morphological changes to BxPC-3 were not observed under themicroscope, even in the presence of AT-264. Furthermore, the RT-PCRresults showed that PepT2 expression was greater than PepT1 expressionin BxPC-3. Accordingly, cell growth inhibition by AT-264 was consideredto be due to the inhibition of PepT2 function, and not due tocytotoxicity. Therefore, it is clear that substances which inhibit thetransport activity of PepT1 or PepT2 also serve as cell growthinhibitors.

Reference Example 5 Production of gp64 Transgenic Mice

gp64 transgenic mice were produced according to the method described inInternational Patent Application No. WO 03/104453. Specifically, themice were produced by the procedures described below.

1) Construction of the gp64 Transgenic Vector

PCR was performed using the gp64 gene sequence (GenBank Acc. No.9627742) as a template, under the conditions described below. As a 5′primer, 64F1 which comprises an EcoRI recognition sequence and a KOZAKsequence at the 5′ end was used (FIG. 6); and as a 3′ primer, 64R1 whichcomprises an EcoRI recognition sequence at the 5′ end was used (FIG. 7).

The PCR reaction solution had the following composition:

5 μL of 10× ExTaq buffer; 4 μL of dNTP that comes with ExTaq; 1 μL of64F1 (10 μmol/L); 1 μL of 64R1 (10 μmol/L); 1 μL of pBac-N-blue (500pg/μL); 0.5 μL of ExTaq (5 unit/μL); and 37.5 μL of diw. The reactionsequence was as follows:94° C. 5 min→(94° C. 15 sec, 57° C. 30 sec, and 72° C. 30 sec)×25 cycles→72° C. 7 min→4° C. (forever)

The amplified band was subcloned into pGEM-T easy, and was used totransform E. coli DH5α. Colony PCR was performed using the T7 and SP6primers. Then, nucleotide sequences of the clones that have beenconfirmed to comprise inserts were analyzed on the ABI Prism 377 DNAsequencer, using the BigDye Cycle Sequence Kit and T7 primer or SP6primer to identify a clone comprising the gene of interest. After theclone was confirmed not to contain any mutations in its nucleotidesequence, a gp64-comprising fragment was excised from this clone usingEcoRI. For transformation of E. coli DH5α, this fragment was insertedinto pCAGGS1 which also had been cleaved with EcoRI. The produced clone,which was as designed, was cultured overnight in 250 mL of LB medium at37° C., and purified using the Endofree MAXI Kit to yield 581.6 μg ofplasmid.

2) Gene Introduction

The DNA fragment to be injected was prepared as described below. First,the gp64 gene-harboring pCAGGS vector (pCAG-gp64; FIG. 8) was treatedwith SalI and PstI to excise a gp64 gene-comprising fragment(approximately 3.8 kb). This fragment (approximately 3.8 kb) wasrecovered using the Gel Extraction Kit (QIAGEN), and then diluted to 3ng/μL with PBS to prepare the DNA fragment to be injected.

Mouse pronuclear stage eggs, to which the DNA fragment was injected,were collected as described below. Superovulation treatment was carriedout by intraperitoneally administering 5 i.u. of PMSG to female Balb/cmice (Clea Japan) initially, and then intraperitoneally administering 5i.u. of hCG 48 hours later. These female mice were mated with male miceof the same strain. Mating was confirmed by the presence of a plug thefollowing morning, and the oviducts of mice were perfused to collect themouse pronuclear stage eggs.

The DNA fragment to be injected was injected into pronuclear stage eggsusing a micromanipulator (Modern Techniques in Gene Targeting (Yodosha),190-207, 2000). The day after the DNA fragment was injected into 373BALB/c embryos, 216 embryos that had developed to the two-cell stagewere transplanted into day 1 pseudopregnant recipient females,approximately ten embryos per oviduct (20 embryos per individual).

Reference Example 6 Preparation of PepT1-Expressing Budding Baculovirus(PepT1-BV)

The PepT1-expressing budding baculovirus to be used as the immunogen wasprepared as described below. PepT1 is a transporter which functions as amembrane protein. The structure of PepT1 is well known (GenBankXM_(—)007063; J. Biol. Chem. 270(12): 6456-6463 (1995)).

A full-length PepT1 gene was isolated from a human renal library usingPCR. The transfer vector, pBlueBacHis-PepT1, was prepared by insertingthe full-length human PepT1 gene into pBlueBacHis2A (Invitrogen). Usingthe Bac-N-Blue Transfection Kit (Invitrogen), the transfer vector wasthen introduced into Sf9 cells, along with a Bac-N-Blue DNA, to producerecombinant viruses for human PepT1 expression. Specifically, 4 μg ofpBlueBacHis-PepT1 was added to the Bac-N-Blue DNA, and then 1 mL ofGrace's medium (GIBCO) and 20 μL of the Cell PECTIN reagent were added,followed by mixing. The mixture was left to stand at room temperaturefor 15 minutes, and then added dropwise to 2×10⁶ Sf9 cells, which hadbeen pre-washed once with Grace's medium. After letting the cells standat room temperature for four hours, an additional 2 mL of completemedium (Grace's medium containing 10% fetal bovine serum (Sigma), 100units/mL of penicillin, and 100 μg/mL streptomycin (GIBCO-BRL)) wasadded, and the cells were then incubated at 27° C. Recombinant virusesfor human PepT1 expression were then produced by homologousrecombination, and purified twice according to the instructions attachedto the kit, to obtain a virus stock of the recombinant virus.

Budding viruses which express human PepT1 were prepared as describedbelow. The recombinant virus thus prepared was used to infect 500 mL ofSf9 cells (2×10⁶/mL) at MOI=5. After culturing at 27° C. for three days,the cultured medium was centrifuged at 800×g for 15 minutes, and thecells and cell debris were removed. The supernatant collected bycentrifugation was further centrifuged at 45,000×g for 30 minutes. Theobtained precipitate were suspended in PBS, and then centrifuged at800×g for 15 minutes to remove the cell components. The budding virusfraction was obtained by centrifuging the supernatant at 45,000×g foranother 30 minutes, and then resuspending the precipitate in PBS.

INDUSTRIAL APPLICABILITY

The present inventors discovered that antibodies which bind to PepT havethe ability to inhibit the transport activity of peptide transporters.These antibodies may be utilized as cell growth inhibitors, for example,for treating or preventing cancer.

1. An isolated antibody, or fragment thereof, that (a) binds to apeptide transporter and (b) inhibits peptide uptake into a cellexpressing the peptide transporter, wherein the antibody is a monoclonalor genetically engineered recombinant antibody, or antigen-bindingfragment thereof.
 2. The antibody or fragment thereof of claim 1,wherein the peptide transporter is a proton motive force (PMF) dependenttransporter.
 3. The antibody or fragment thereof of claim 1, wherein thepeptide transporter is PepT1.
 4. The antibody or fragment thereof ofclaim 1, wherein the peptide transporter is PepT2.
 5. The antibody orfragment thereof of claim 1, wherein the antibody is monoclonal.
 6. Theantibody or fragment thereof of claim 1, wherein the antibody orfragment thereof is recombinant.
 7. The antibody or fragment thereof ofclaim 1, wherein the antibody is humanized or chimeric.
 8. The antibodyor fragment thereof of claim 1, wherein the antibody is bispecific. 9.An isolated antibody, or fragment thereof, that (a) binds to a peptidetransporter and (b) inhibits the growth of a cell, wherein the antibodyis a monoclonal or genetically engineered recombinant antibody, orantigen-binding fragment thereof.
 10. The antibody or fragment thereofof claim 9, wherein the peptide transporter is a PMF dependenttransporter.
 11. The antibody or fragment thereof of claim 9, whereinthe peptide transporter is PepT1.
 12. The antibody or fragment thereofof claim 9, wherein the peptide transporter is PepT2.
 13. The antibodyor fragment thereof of claim 9, wherein the cell is a cancer cell. 14.The antibody or fragment thereof of claim 9, wherein the cell is apancreatic cancer cell.
 15. The antibody or fragment thereof of claim 9,wherein the antibody is monoclonal.
 16. The antibody or fragment thereofof claim 9, wherein the antibody or fragment thereof is recombinant. 17.The antibody or fragment thereof of claim 9, wherein the antibody ishumanized or chimeric.
 18. The antibody or fragment thereof of claim 9,wherein the antibody is bispecific.
 19. A diabody that binds to apeptide transporter and inhibits peptide uptake into a cell expressingthe peptide transporter.
 20. The diabody of claim 19, wherein thepeptide transporter is a PMF dependent transporter.
 21. The diabody ofclaim 19, wherein the peptide transporter is PepT1.
 22. The diabody ofclaim 9, wherein the peptide transporter is PepT2.
 23. A compositioncomprising the antibody or fragment thereof of claim 1 and apharmaceutically acceptable carrier, wherein the antibody or fragmentthereof inhibits the growth of a cell.
 24. The composition of claim 23,wherein the cell is a cancer cell.
 25. The composition of claim 23,wherein the cell is a pancreatic cancer cell.
 26. A compositioncomprising the antibody or fragment thereof of claim 9 and apharmaceutically acceptable carrier, wherein the antibody or fragmentthereof inhibits the growth of a cell.
 27. The composition of claim 26,wherein the cell is a cancer cell.
 28. The composition of claim 26,wherein the cell is a pancreatic cancer cell.